Immunotherapies that utilize cytotoxic T lymphocytes (CTLs) have proven effective at eradicating large tumor burdens in both animal models and human cancer patients. Since dendritic cells (DCs) are the most potent antigen-presenting cells for priming naive CD8+ T-cells to become activated CTLs that efficiently kill target cells in an MHC class I-restricted fashion, there has been widespread interest in developing DC-based vaccines for use in cancer therapy. The specific objective of this project is to generate an improved DC vaccine by exploiting the natural mechanisms that control MHC-I trafficking and DC surface expression to improve the quality and duration of tumor antigen presentation to CD8+ T cells. It is our central hypothesis that conserved motifs within the MHC-I cytoplasmic tail control not only the duration of presentation of MHC-I/peptide complexes at the cell surface, but also MHC-I trafficking through specialized, endocytic cross-presentation compartments. We have formulated this hypothesis on the basis of our Preliminary Results identifying two functionally distinct MHC-I tail motifs that directly control DC endocytic trafficking and cross-presentation function of murine MHC-I molecules, and which play a crucial role in the generation of antiviral CTL responses in vivo (Lizee et al, Nature Immunology). The rationale for this proposal is that utilizing our knowledge of how these conserved motifs operate in DCs will allow for the ability to improve tumor antigen loading and extend duration of antigen presentation in human DC-based cancer vaccines, thus improving CTL priming outcomes. We plan to test our central hypothesis and accomplish our overall objective of improving DC-based cancer vaccines by focusing on the following three specific aims: (1) Determine how modifications to the MHC-I cytoplasmic tail impact the priming of antigen-specific CTLs and alter the dynamics of DC antigen presentation. (2) Using established murine models, assess the efficacy of MHC-I tail-modified DC vaccines in priming antigen-specific CTLs and in the induction of antitumor responses. (3) Analyze how inflammatory mediators and tumor-derived factors affect MHC-I tail phosphorylation, intracellular trafficking, and antigen presentation in DCs. The proposed work is innovative, because it will uncover the molecular mechanisms utilized by DCs to prime optimally effective antitumor CTL responses. It will also fill in gaps in the current knowledge base with regard to the dynamic changes in MHC-I trafficking and antigen presentation that occur during DC activation by toll-like receptor (TLR)-ligands or innate immune signals. Such results will have an important positive impact, because they will pave the way towards the next generation of improved, DC-based vaccines. They will enable the design of novel therapeutics capable of modifying the MHC-I tail, thus potentially allowing for manipulation of immune responses at the level of MHC-I antigen presentation. Successful completion of these studies is likely to have an impact in other areas of human disease treatment, including autoimmunity, transplant immunology, and pathogen infections. PUBLIC HEALTH RELEVANCE: Cancer vaccines work by eliciting specific cytotoxic ('killer') T lymphocytes (CTL) in the blood of cancer patients, which in turn destroy the tumor. Dendritic cell (DC)-based vaccines have shown much promise clinically, but our lack of basic understanding of DC biology has limited the development of these vaccines. This grant proposes to utilize our basic knowledge of MHC class I biology to improve the potency of DC-based based cancer vaccines, with the ultimate goal of improving patient responses to CTL-based immunotherapy.